Molecular display system

ABSTRACT

There is provided herein a method for identifying and/or recovering at least one genetically encoded affinity reagent specific for a target molecule by screening using molecular display in conjunction with the sequencing of positive and negative selection pools from the screen.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/344,971, filed Nov. 7, 2016 and issued as U.S. patent Ser. No.10,746,743 on Aug. 18, 2020, which is a continuation of U.S. patentapplication Ser. No. 13/629,520, filed Sep. 27, 2012, now abandoned,which claims the benefit of provisional application U.S. Ser. No.61/539,546 filed Sep. 27, 2011, the contents of which are hereinincorporated by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing“P56672US01_SequenceListing” (75,596 bytes) created on Aug. 11, 2020 ishereby incorporated by reference.

FIELD OF INVENTION

This invention relates to the field of screening for affinity reagentsto a molecular target, and more specifically to molecular displaymethods used in conjunction with sequencing.

BACKGROUND

Molecular display technologies are widely used to screen for potentialaffinity binders to a specific target molecule, however, there ispotential for improving thereon. For example, phage display antibodytechnologies are used for isolating antibody fragments specific toantigens of interest, but selection of libraries against cell-surfaceantigens remains very challenging. The heterogeneity of the cell-surfaceand, accordingly, the relatively low concentration of the targetantigen, give rise to large numbers of background phage clones. Thesephage clones may be non-specific binding clones, or may be specific forantigens other than the desired cell-surface target. Consequently, poorenrichment for binding phage clones is typically observed in cellselections. However, many proteins require the membrane environment forproper folding and stability and, as such, the ability to selectphage-displayed antibody libraries against cell-surface epitopes remainscrucial. If a protein is not properly folded, certain epitopes may notbe available for binding by, for example, an affinity reagent. Likewise,proteins that are part of large complexes or associated with DNA,histones or other subcellular structures contain epitopes that are notnecessarily made available for binding following traditionalpurification methods. For example, the properties of multi-pass membraneG-protein coupled receptors make their expression and purification verydifficult, yet they are particularly relevant drug targets [1,2].Indeed, the high specificity of monoclonal antibodies, combined withtheir ability to engage immune mechanisms, makes this class of biologicsof particular interest in the treatment of numerous cancers andinfectious diseases [3,4,5]. A reliable selection methodology fortargeting exposed epitopes (e.g. cell-surface epitopes), whicheliminates the need for highly purified antigens, would significantlyexpand the range of antigens that could be targeted by therapeuticmonoclonal antibodies.

Phage display selection strategies to reduce background binding to cellshave included negative or competitive pre-absorption steps againstmultiple cell-lines [6,7,8,9,10] and various strategies to removeunbound from bound phage, including centrifugation through a densitygradient [11,12] and the pathfinder approach [13,14]. Although thesemethods may help to enrich for phage clones specific to the antigen ofinterest, the number of unique antibody fragments recovered by thesemethods often remains relatively low, as phage display methodologiestypically exhibit an affinity based selection pressure that promotessequence convergence in later rounds of selection. New strategies arerequired to identify less prevalent clones that may exhibit desirablebinding properties.

SUMMARY OF THE INVENTION

The methods described herein provide a rapid, efficient method ofidentifying binding agents, e.g., antibodies and antigen-bindingfragments thereof, that specifically bind to cell-surface targets andother cell-surface expressed antigens. These methods include deepsequencing/high-throughput sequencing followed by a recovery method,also referred to herein as a rescue strategy. As used herein, the term“deep sequencing” and variations thereof refers to the number of times anucleotide is read during the sequencing process. Deep sequencingindicates that the coverage, or depth, of the process is many timeslarger than the length of the sequence under study. Suitable deepsequencing methods include the methods described herein or any otherart-recognized techniques. Suitable rescue strategies include the clonalELISA assays and PCR rescue strategies described herein or any otherart-recognized techniques. The methods provided herein do not requireadditional purification and/or isolation steps prior to identificationand recovery of the binding agent, e.g., antibody or antigen-bindingfragment thereof.

The methods provided herein are useful in identifying binding agents,e.g., antibodies and antigen-binding fragments thereof, which are nothighly expressed in a given display. For example, the methods providedherein are useful in identifying polypeptide sequences that compriseless than 25%, less than 20%, less than 15%, less than 10%, less than5%, less than 4%, less than 3%, less than 2%, less than 1%, less than0.5% and/or less than 0.25% of the selection pool.

The methods provided herein are useful in differential selectionstrategies, for example, to identify binding agents that bind a givencell-surface target only when the target exhibits a particularmodification, a particular conformation or other identifyingcharacteristic. The methods provided herein are also useful indifferential selection strategies, for example, to identify bindingagents that bind a given cell-surface target only under certainmetabolic or other biological conditions. The methods provided hereinare also useful in differential selection strategies, for example, toidentify binding agents that bind a given cell-surface target only inthe presence of an effector, a target-binding partner or other moleculethat must be present to enable binding between the genetically encodedbinding agent and the target.

The methods provided herein are useful for identifying binding agents,particularly, binding polypeptides including antibodies andantigen-binding fragments thereof, also referred to herein asimmunologically active fragments. In some embodiments, the antibody orantigen-binding fragment thereof is a monoclonal antibody, domainantibody, single chain, Fab fragment, a F(ab′)₂ fragment, a scFv, ascab, a dAb, a single domain heavy chain antibody, and a single domainlight chain antibody. In some embodiments, such an antibody orimmunologically active fragment thereof that binds a given antigen,e.g., a cell-surface target, is a mouse, chimeric, humanized or fullyhuman monoclonal antibody.

In some embodiments, the cell-surface target is selected from the groupconsisting of HER2, CD133, ErbB3, Fzd7, ROR1, ROR2, exon16 deletedErbB2, and ITGA11. In some embodiments, the cell-surface target includesa modification that is required for epitope binding, such as, forexample, an O-linked N-acetylglucosamine (O-GlcNAc) modification.

These cell-surface targets are expressed on mammalian cells. Suitablemammalian cells for use in the methods provided herein include, but arenot limited to, cells such as 293, 293T, C2C12, and/or MC7 cells.

The methods provided herein are used in combination with phage-displaylibraries referred to herein as Libraries F and G, but those of ordinaryskill in the art will appreciate that these methods can be used inconjunction with any peptide/polypeptide display system in whichcell-surface targets/antigens are expressed. Library G is an scFv-phagelibrary that was constructed by introducing degenerate codons intopositions in CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 of asingle human ScFv framework. Library F is an Fab-phage library that wasconstructed by introducing degenerate codons into positions in CDR-H1,CDR-H2, CDR-H3 and CDR-L3 of a single human Fab framework. Library F wasconstructed using an anti-maltose binding protein Fab as a template.

In an aspect, there is provided a method for identifying and/orrecovering at least one genetically encoded affinity reagent specificfor a target molecule, the method comprising: providing a moleculardisplay system which displays a library of potential genetically encodedaffinity reagents; screening the library against the target molecule toproduce positive and negative selection pools; sequencing geneticallyencoded affinity reagents in each of the positive and negative selectionpools; identifying at least one sequence that is more abundant in thepositive selection pool as compared to the negative selection pool; andrecovering at least one clone corresponding to the sequence.

In a further aspect, there is provided an antibody or antibody fragmentcomprising any one of CDR regions outlined in FIG. 2 , FIG. 5 or FIG. 9. Preferably, the antibody or antibody fragment is selected from thegroup consisting of antibodies or antibody fragments comprising CDRL3,CDRH1, CDRH2 and CDRH3 of any one of clones WY574B, WY574E, WY574F,WY677C and WY677D described herein, the CDRH3 regions shown in FIG. 5 orthe combinations of CDRL3 and CDRH3 regions shown in FIG. 9 . In oneembodiment, the antibody or antibody fragment is useful for thetreatment of cancer, e.g., Her-2 positive cancer, preferably selectedfrom the group consisting of breast cancer, ovarian cancer, uterinecancer and stomach cancer.

The invention provides antibodies and antigen-binding fragments thereofthat bind HER2 and include a variable heavy chain complementaritydetermining region 1 (CDR-H1) comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 18, 22, 26, 30 and 34; avariable heavy chain complementarity determining region 2 (CDR-H2)comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 19, 23, 27, 31 and 35; a variable heavy chaincomplementarity determining region 3 (CDR-H3) comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 20, 24, 28,32 and 36. In some embodiments, these anti-HER2 antibodies andantigen-binding fragments thereof also include a variable light chaincomplementarity determining region 1 (CDR-L1) comprising the amino acidsequence SVSSA (SEQ ID NO: 240); a variable light chain complementaritydetermining region 2 (CDR-L2) comprising the amino acid sequence SASSLYS(SEQ ID NO: 241); and a variable light chain complementarity determiningregion 3 (CDR-L3) comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 17, 21, 25, 29 and 33.

The invention provides antibodies and antigen-binding fragments thereofthat bind HER2 and include a CDR-L1 comprising the amino acid sequenceSVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acid sequenceSASSLYS (SEQ ID NO: 241), a CDR-L3 comprising the amino acid sequenceX₁-X₂-X₃-X₄-X₅-X₆ (SEQ ID NO: 242), where X₁, X₂, X₃, and X₄ are Y, S,G, A, F, W, H, P or V and X₅ is P or L and X₆ is I or L; a CDR-H1comprising the amino acid sequence X₁-X₂-X₃-X₄-X₅-X₅ (SEQ ID NO: 243),where X₁ is I or L, X₂, X₃, X₄, and X₅ are Y or S and where X₆ is I orM; and a CDR-H2 comprising the amino acid sequenceX₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉ (SEQ ID NO: 244), where X₁, X₂, X₄, X₅,X₅, X₅, and X₉ is Y or S, X₃ is P or S, and where X₇ is G or S; and aCDR-H3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 37-136.

The invention provides antibodies and antigen-binding fragments thereofthat bind CD133 and include a CDR-L1 comprising the amino acid sequencethe amino acid sequence Q-X₁-X₂-X₃-X₄-X₅ (SEQ ID NO: 245), where X₁, X₂,X₃, X₄, and X₅ are Y, S or, G; a CDR-L2 comprising the amino acidsequence X₁-A-S—X₂-L-Y (SEQ ID NO: 246), where X₁ and X₂ are Y, S or, G;a CDR-L3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157 and 159; a CDR-H1 that includes the amino acid sequenceX₁-X₂-X₃-X₄-X₅-X₆ (SEQ ID NO: 247), where X₁ is I or L, X₂, X₃, X₄, andX₅ are Y, S or G and where X₆ is I or M; a CDR-H2 that includes theamino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉ (SEQ ID NO: 266),where X₁, X₂, X₄, X₅, X₆, X₈, and X₉ is Y, S or G, X₃ is P or S, andwhere X₇ is G or S; and a CDR-H3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158 and 160.

The invention provides antibodies and antigen-binding fragments thereofthat bind ErbB3 and include a CDR-L1 comprising the amino acid sequencethe amino acid sequence Q-X₁-X₂-X₃-X₄-X₅ (SEQ ID NO: 245), where X₁, X₂,X₃, X₄, and X₅ are Y, S or, G; a CDR-L2 comprising the amino acidsequence X₁-A-S—X₂-L-Y (SEQ ID NO: 246), where X₁ and X₂ are Y, S or, G;a CDR-L3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 161, 163, 165, 167, 169, 171, 173 and 175; aCDR-H1 that includes the amino acid sequence X₁-X₂-X₃-X₄-X₅-X₆ (SEQ IDNO: 247), where X₁ is I or L, X₂, X₃, X₄, and X₅ are Y, S or G and whereX₆ is I or M; a CDR-H2 that includes the amino acid sequence (SEQ ID NO:266), where X₁, X₂, X₄, X₅, X₆, X₈, and X₉ is Y, S or G, X₃ is P or S,and where X₇ is G or S; and a CDR-H3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 162, 164, 166, 168,170, 172, 174 and 176.

The invention provides antibodies and antigen-binding fragments thereofthat bind Fzd7 and include a CDR-L1 comprising the amino acid sequenceSVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acid sequenceSASSLYS (SEQ ID NO: 241), a CDR-L3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 177, 179 and 181; aCDR-H1 comprising the amino acid sequence X₁-X₂-X₃-X₄-X₅-X₆ (SEQ ID NO:243), where X₁ is I or L, X₂, X₃, X₄, and X₅ are Y or S and where X₆ isI or M; and a CDR-H2 comprising the amino acid sequenceX₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉ (SEQ ID NO: 244), where X₁, X₂, X₄, X₅,X₆, X₈, and X₉ is Y or S, X₃ is P or S, and where X₇ is G or S; and aCDR-H3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 178, 180 and 182.

The invention provides antibodies and antigen-binding fragments thereofthat bind ROR1 and include a CDR-L1 comprising the amino acid sequenceSVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acid sequenceSASSLYS (SEQ ID NO: 241), a CDR-L3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 183, 185 and 187; aCDR-H1 comprising the amino acid sequence X₁-X₂-X₃-X₄-X₅-X₆ (SEQ ID NO:243), where X₁ is I or L, X₂, X₃, X₄, and X₅ are Y or S and where X₆ isI or M; and a CDR-H2 comprising the amino acid sequenceX₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉ (SEQ ID NO: 244), where X₁, X₂, X₄, X₅,X₆, X₈, and X₉ is Y or S, X₃ is P or S, and where X₇ is G or S; and aCDR-H3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 184, 186 and 188.

The invention provides antibodies and antigen-binding fragments thereofthat bind ROR2 and include a CDR-L1 comprising the amino acid sequenceSVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acid sequenceSASSLYS (SEQ ID NO: 241), a CDR-L3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 189, 191, 193, 195,197 and 199; a CDR-H1 comprising the amino acid sequenceX₁-X₂-X₃-X₄-X₅-X₆ (SEQ ID NO: 243), where X₁ is I or L, X₂, X₃, X₄, andX₅ are Y or S and where X₆ is I or M; and a CDR-H2 comprising the aminoacid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉ (SEQ ID NO: 244), where X₁,X₂, X₄, X₅, X₆, X₈, and X₉ is Y or S, X₃ is P or S, and where X₇ is G orS; and a CDR-H3 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 190, 192, 194, 196, 198 and 200.

The invention provides antibodies and antigen-binding fragments thereofthat bind an ErbB2 variant known as exon 16 deleted ErbB2 and include aCDR-L1 comprising the amino acid sequence SVSSA (SEQ ID NO: 240), aCDR-L2 that includes the amino acid sequence SASSLYS (SEQ ID NO: 241), aCDR-L3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 201, 203 and 205; a CDR-H1 comprising theamino acid sequence X₁-X₂-X₃-X₄-X₅-X₆ (SEQ ID NO: 243), where X₁ is I orL, X₂, X₃, X₄, and X₅ are Y or S and where X₆ is I or M; and a CDR-H2comprising the amino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉ (SEQID NO: 244), where X₁, X₂, X₄, X₅, X₆, X₈, and X₉ is Y or S, X₃ is P orS, and where X₇ is G or S; and a CDR-H3 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 202, 204 and206.

The invention provides antibodies and antigen-binding fragments thereofthat bind ITGA11 and include a CDR-L1 comprising the amino acid sequenceSVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acid sequenceSASSLYS (SEQ ID NO: 241), a CDR-L3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 207, 209, 211 and 213;a CDR-H1 comprising the amino acid sequence X₁-X₂-X₃-X₄-X₅-X₆ (SEQ IDNO: 243), where X₁ is I or L, X₂, X₃, X₄, and X₅ are Y or S and where X₆is I or M; and a CDR-H2 comprising the amino acid sequenceX₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉ (SEQ ID NO: 244), where X₁, X₂, X₄, X₅,X₆, X₈, and X₉ is Y or S, X₃ is P or S, and where X₇ is G or S; and aCDR-H3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 208, 210, 212 and 214.

The invention provides antibodies and antigen-binding fragments thereofthat recognize a modification known as O-GlcNac modification and includea CDR-L1 comprising the amino acid sequence SVSSA (SEQ ID NO: 240), aCDR-L2 that includes the amino acid sequence SASSLYS (SEQ ID NO: 241), aCDR-L3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 215, 217, 219 and 221; a CDR-H1 comprising theamino acid sequence X₁-X₂-X₃-X₄-X₅-X₆ (SEQ ID NO: 243), where X₁ is I orL, X₂, X₃, X₄, and X₅ are Y or S and where X₆ is I or M; and a CDR-H2comprising the amino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉ (SEQID NO: 244), where X₁, X₂, X₄, X₅, X₆, X₈, and X₉ is Y or S, X₃ is P orS, and where X₇ is G or S; and a CDR-H3 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 216, 218, 220and 222.

In a further aspect, there is provided a method of treating cancer,e.g., Her-2 positive cancer, preferably selected from the groupconsisting of breast cancer, ovarian cancer, uterine cancer and stomachcancer, in a patient comprising administering to the patient atherapeutically effective amount of the antibody or antibody fragmentdescribed herein.

In a further aspect, there is provided a method of treating a disorderthat is associated with aberrant expression and/or activity of thecell-surface target against which the antibody has been selected,comprising administering to the patient a therapeutically effectiveamount of the antibody or antibody fragment described herein.

In a further aspect, there is provided a use of the antibody or antibodyfragment described herein for the treatment of cancer, e.g., Her-2positive cancer, preferably selected from the group consisting of breastcancer, ovarian cancer, uterine cancer and stomach cancer.

In a further aspect, there is provided a use of the antibody or antibodyfragment described herein for the treatment of a disorder that isassociated with aberrant expression and/or activity of the cell-surfacetarget against which the antibody has been selected.

In a further aspect, there is provided a use of the antibody or antibodyfragment described herein in the preparation of a medicament for thetreatment of Her-2 positive cancer, preferably selected from the groupconsisting of breast cancer, ovarian cancer, uterine cancer and stomachcancer.

In a further aspect, there is provided a use of the antibody or antibodyfragment described herein in the preparation of a medicament for thetreatment of a disorder that is associated with aberrant expressionand/or activity of the cell-surface target against which the antibodyhas been selected.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments of the invention may best be understood by referring to thefollowing description and accompanying drawings. In the description anddrawings, like numerals refer to like structures or processes. In thedrawings:

FIG. 1A is a flow chart of the selection strategy used to isolate Fabclones specific for cell-surface displayed Her2. The positive selectionbegins a pre-absorption step in which the library phage are incubatedwith untransfected 293T cells. After incubation, the mixture is pelletedto remove the library clones bound to the cells. These clones are likelyspecific for cell-surface epitopes that are not of interest, or arenon-specific binding clones. The phage of interest for subsequent stepsare circled in red. The library phage remaining in the supernatant areincubated with the Her2 transfected 293T cells, non-binding phage arewashed away, and the phage bound to the transfected cells are amplifiedin an E. coli host. The amplified phage are then purified and used inthe next round of selection. In parallel, the negative selection iscarried out by incubating library phage with untransfected 293T cells.Phage clones that do not bind to the cells are washed away, and theremaining bound phage (circled in red) are amplified in an E. coli hostfor the next round of selection. FIG. 1B shows Phage clones beingrescued from the positive selection output pool using a PCR strategy inwhich abutting forward and reverse PCR primers (shown in red) anneal tothe unique heavy chain CDR3 sequence (represented by the colored portionof the circular DNA molecules). PCR amplification results in synthesisof the complete phagemid vector. Ligation of the PCR product yieldsclosed, circular, double-stranded DNA that can be transformed intobacteria for rescue. Dpn1 digestion of the PCR product degradesundesired phagemid DNA present from the positive selection pool(represented in gray), due to the presence of methylated Dpn1recognition sites. As a consequence of in vitro synthesis, the PCRproduct is not methylated and, therefore, is not recognized by Dpn1.

FIG. 2 shows Her2 specific clones rescued from the positive selectionpool. Five clones were rescued from the Her2 positive selection outputpool by PCR amplification with primers specific to their unique CDR H3sequence. The total number of times each CDRH3 was observed in thepositive and negative selection pools is listed. The abundance of eachsequence in the positive pool is also listed, as a percentage of thetotal number of sequences isolated. The CDR loops are defined by theIMGT nomenclature (Lefranc, Pommie, Ruiz et al (2003) Dev Comp Immunol27, 55-77).

FIG. 3A shows the analysis of Fab binding to cell-surface Her2 by flowcytometry. Fabs WY574B (left panel), WY574E (middle panel), and WY 547F(right panel) were tested for binding to Her2 and EGFR transfected 293Tcells. Binding of the anti-Her2 Fab proteins was detected using anAlexa488-conjugated secondary antibody (AF488) against a Flag-epitope onthe C terminus of the Fab light-chain. The stained anti-Her2 transfectedpopulation is shown in green, the stained EGFR transfected population isshown in blue, and the unstained Her2 transfected population is shown ingray. The AF-488 positive cell gate is indicated. FIG. 3B shows that theFabs were also testing for binding to Her2 positive (BT474) and negative(T47D) human breast cancer cell-lines, using the same secondarydetection as in (a). The stained BT474 population is shown in green inthe bottom panel, with the unstained population shown in gray. Thestained T47D cell population is indicated in blue.

FIG. 4A and FIG. 4B show the binding specificity of synthetic anti-HER2antibodies against live cells. (FIG. 4A) 293T cells were seeded oncoverslips coated with 50 mg/ml poly-D-lysine for 24 h followed bytransient transfection of plasmid encoding HER2. (FIG. 4B) BT474 andT47D breast cancer cells were seeded onto uncoated coverslips. 48 hpost-transfection or post-seeding, the cells were fixed with 3.7%formaldehyde without permeabilization and stained with anti-HER2 Fabprotein (5 mg/ml) followed Alexa488-conjugated secondary antibodyagainst a Flag-epitope on the C terminus of the Fab light-chain. Thenuclei were stained using the Hoechst dye. The images were acquiredusing the WaveFX spinning disk confocal microscope by QuoromTechnologies Inc. Composite images of the ‘xy’ and ‘yz’ planes arerepresented (scale bar, 10 um).

FIG. 5 shows the 100 most frequent CDRH3 sequences obtained fromIllumina sequencing of the positive selection pool. The 100 mostfrequently observed CDR H3 sequences (positions 107-117 per IMGT)obtained from the round 3 positive selection output are listed, startingwith the most frequently observed sequence. The number of countsreflects the number of times each sequence was observed in the positiveor negative selection pool, or in the unselected naïve library.Sequences highlighted in yellow represent those clones that were rescuedfrom the positive selection pool. Sequence number 13 corresponds to thewild type sequence that was used as the template in the libraryconstruction process.

FIG. 6A and FIG. 6B show rescue strategies that utilize both the uniqueheavy chain CDR3 (CDRH3) sequence and light chain CDR3 (CDRL3) sequencesidentified using the methods provided herein. (FIG. 6A) Two primer setsspecific for both CDRH3 and CDRL3 are used to make recovery morespecific. (FIG. 6B) Three primer sets are used to amplify threefragments in a strategy that makes use of both the CDRH3 and CDRL3sequences, as well as unique Nsi1 and Nhe1 restriction sites in thelibrary phage vector.

FIG. 7 is a flow chart of the selection strategy used to isolate Fabclones specific for cell surface O-GlcNAc-dependent epitopes.

FIG. 8 is an ELISA graph of binders identified from the selectionstrategy used to identify Fab clones specific for cell surfaceO-GlcNAc-dependent epitopes.

FIG. 9 shows the phage-Fab clones that were rescued from the positiveselection pool.

FIG. 10 shows deep sequencing strategies to decode variablecomplementarity determining regions (CDRs) in pools of syntheticantibody fragments. The region of the phagemid encoding the Fab scaffold(solid black line) and its six CDRs (white boxes labeled L1, L2, L3, H1,H2, H3) is shown. PCR primers to generate amplicon sequencing librariesare shown as solid black arrows. Sequencing read orientations are shownas white block arrows. Strategies 1 and 2 are compatible with Illuminaplatforms and decode two or more CDRs. Strategy 3 is compatible withIonTorrent platforms and decodes only CDR-H3.

DETAILED DESCRIPTION

There is described herein the development of a new method for selectingfor affinity reagents against a target molecule.

In a specific example, there is described a new method for selectingphage display libraries against cell-surface expressed antigens. Thismethodology, termed CellectSeq, combines the use of phage-displayedsynthetic antibody libraries and high throughput DNA sequencingtechnology. In the synthetic library approach, the antigen binding sitecontains ‘man-made’ diversity, which is introduced into human frameworkregions based on existing knowledge of antibody structure and function[15]. Consequently, synthetic libraries can be biased towards antibodyclones with favorable properties, such as high stability and expression.The use of high throughput DNA sequencing enables the rapididentification of high affinity clones specific to cells that expressthe antigen of interest. Moreover, the methodology we report here allowsrare binding clones, which may compose as little as 0.25% of theselection pool, to be identified and successfully rescued.

As an initial model system, we selected synthetic antibody librariesagainst cells transiently transfected to express the human epidermalgrowth factor receptor 2 (Her2, also known as ErbB2). A member of thehuman epidermal growth factor receptor (EGFR) family, Her2 is atransmembrane tyrosine kinase receptor involved in signalling pathwaysthat promote cell proliferation and survival [16,17]. Her2 isoverexpressed in approximately 20 to 25% of invasive breast cancers[18,19], and its overexpression correlates with increased tumoraggressiveness, an increased chance of recurrence, and poor prognosis inbreast cancer patients [20,21]. We selected phage-displayed syntheticantibody libraries against 293T cells transiently transfected to expressHer2 and, in parallel, untransfected 293T cells. After three rounds ofselection, each output pool was subjected to Illumina deep sequencing.We found that comparing the deep sequencing results of the positive andnegative selection pools could identify Her2 specific clones. We wereable to rescue clones unique to the positive selection pool usingprimers specific to the third hypervariable loop of the antibody heavychain (CDR H3), and demonstrated that the rescued clones bindspecifically and with high affinity to our target antigen, Her2. Ourresults suggest that the use of deep sequencing enables efficientidentification of antibody fragments specific to target antigenspresented on cell-surfaces.

While the initial model system used synthetic antibody librariesscreened against cells expressing HER2, it is understood that themethods described herein are useful to identify binding agents thatrecognize any number of targets that are expressed on a cell-surface.

In an aspect, there is provided a method for identifying and/orrecovering at least one genetically encoded affinity reagent specificfor a target molecule, the method comprising: providing a moleculardisplay system which displays a library of potential genetically encodedaffinity reagents; screening the library against the target molecule toproduce positive and negative selection pools, preferably with multiplerounds of selection; sequencing genetically encoded affinity reagents ineach of the positive and negative selection pools; identifying at leastone sequence that is more abundant in the positive selection pool ascompared to the negative selection pool; and recovering at least oneclone corresponding to the sequence.

As used herein, “affinity reagent” is any molecule that specificallybinds to a target molecule, for example, to identify, track, capture orinfluence the activity of the target molecule. The affinity reagentsidentified or recovered by the methods described herein are “geneticallyencoded”, for example an antibody, peptide or nucleic acid, and are thuscapable of being sequenced. As used herein, the terms “protein”,“polypeptide” and “peptide” are used interchangeably to refer to two ormore amino acids linked together.

As used herein, “molecular display system” is any system capable ofpresenting a library of potential affinity reagents to screen forpotential binders to a target molecule or ligand, for example, throughin vitro protein evolution. Examples of display systems include phagedisplay, bacterial display, yeast display, ribosome display and mRNAdisplay. In one embodiment of the method, phage display is used.

In some embodiments, the sequencing is deep/high-throughput sequencing.Examples of deep/high-throughput sequencing include Lynx Therapeutics'Massively Parallel Signature Sequencing (MPSS), Polony sequencing, 454pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, Ionsemiconductor sequencing (Ion Torrent by Life Technologies™), and DNAnanoball sequencing. In a preferable embodiment, Illumina sequencing isused.

In some embodiments, the rescue strategy is a clonal ELISA assay, aPCR-based rescue strategy, including the clonal ELISA assay andPCR-based rescue strategies described herein.

In some embodiments, the affinity reagents are selected from the groupconsisting of nucleic acid molecules and polypeptides. In oneembodiment, the affinity reagents are antibodies, preferably syntheticantibodies, and further preferably the library is a synthetic Fab orscFv library.

In some embodiments, each of the affinity reagents in the librarycontains unique sequence tags and the sequencing identifies the uniquesequence tags. Preferably, the at least one clone is recovered byannealing primers specific for the unique sequence tags. For example, ina preferred embodiment, the library is a synthetic Fab library and theunique sequence tag is in the CDR H3 region.

In some embodiments, the target molecule is a cell surface protein. Infurther embodiments, the screening is performed against the targetmolecule presented on a cell surface. In some embodiments, the screeningis performed against the target molecule presented on a mammalian cellsurface.

In some embodiments, the sequences identified are more abundant in thepositive selection pool as compared to the negative selection pool by afactor of at least 2, and in increasing preferably at least 3, at least4 and at least 5.

The methods provided herein are used in combination with phage-displaylibraries referred to herein as Libraries F and G, but those of ordinaryskill in the art will appreciate that these methods can be used inconjunction with any peptide/polypeptide display system in whichcell-surface targets/antigens are expressed.

Library G is an scFv-phage library that was constructed by introducingdegenerate codons into positions in CDR-H1, CDR-H2, CDR-H3, CDR-L1,CDR-L2 and CDR-L3 of a single human ScFv framework. The library has atotal diversity of 1.08×10¹¹ unique clones, and the details of thelibrary design are shown in Table 1 below, where the bolding in theCDR-L3 and CDR-H3 regions represents positions that were replaced byrandom loops of all possible varying lengths, as indicated.

TABLE 1 CDR Sequences of Library G clones CDR-L1 (SEQ ID NO: 256)Position 27 28 29 30 31 32 Q YSG YSG YSG YSG YSG CDR-L2 (SEQ ID NO: 257)Position 50 51 52 53 54 55 YSG A S YSG L Y CDR-L3 (SEQ ID NO: 258) LoopLength (8-12 aa) Q Q Z Z Z Z PL FI T Z = 25% Y, 20% S, 20% G, 10% A, and5% each of F, W, H, P, V CDR-H1 (SEQ ID NO: 259) Position 29 30 31 32 3334 IL YSG YSG YSG YSG IM CDR-H2 (SEQ ID NO: 260) Position 50 51 52 52a53 54 55 56 57 58 YSG I YSG PS YSG YSG GS YSG T YSG CDR-H3 (SEQ ID NO:261) Loop Length (7-19 aa) Position 98 99 100 101 102 103 104 104a 104b104c 105 R Z Z Z Z Z Z Z AG FILM D Z = 25% Y, 20% S, 20% G, 10% A, and5% each of F, W, H, P, V

The nucleotide sequence of the vector encoding Library G is shown below:

FEATURES Location/Qualifiers rep_origin 3764 . . . 4235/note = “f1 origin” sig_peptide 1534 . . . 1602/note = “ST2 secr signal” promoter 21 . . . 52 /note = “LacIq promoter”promoter 1412 . . . 1439 /note = “pTac promoter” ORF 87 . . . 1169/note = “LacIg” ORF complement(5461 . . . 6321) /note = “AmpR” ORF2416 . . . 2880 /note = “III gene (truncated)” misc_feature2008 . . . 2385 /note = “VH” misc_feature 1639 . . . 1959 /note = “VL”misc_feature 1960 . . . 2007 /note = “linker C3” misc_feature2302 . . . 2346 /note = “CDRH3” misc_feature 2155 . . . 2184/note = “CDRH2” misc_feature 1606 . . . 1629 /note = “FLAG tag”misc_feature 1786 . . . 1806 /note = “CDRL2” misc_feature2092 . . . 2109 /note = “CDRH1” misc_feature 1909 . . . 1926/note = “CDRL3” misc_feature 2389 . . . 2403 /note = “hinge”misc_feature 1720 . . . 1734 /note = “CDRL1” misc_feature2404 . . . 2415 /note = “dimerization domain”   1 gaattcccga caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga tagcgcccgg  61 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt 121 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg 181 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg 241 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg 301 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg 361 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc 421 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg 481 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg 541 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg 601 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg 661 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc 721 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa 781 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg 841 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg 901 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg 961 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg1021 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc1081 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac1141 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact1201 cattaggcac aattctcatg tttgacagct tatcatcgac tgcacggtgc accaatgctt1261 ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat1321 aattcgtgtc gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata1381 acggttctgg caaatattct gaaatgagct gttgacaatt aatcatcggc tcgtataatg1441 tgtggaattg tgagcggata acaatttcac acaggaaaca gccagtccgt ttaggtgttt1501 tcacgagcac ttcaccaaca aggaccatag attatgaaaa agaatatcgc atttcttctt1561 gcatctatgt tcgttttttc tattgctaca aatgcctatg catccgatta caaagatgac1621 gatgacaaag gcggtggcga tatccagatg acccagtccc cgagctccct gtccgcctct1681 gtgggcgata gggtcaccat cacctgccgt gccagtcagt ccgtgtccag cgctgtagcc1741 tggtatcaac agaaaccagg aaaagctccg aagcttctga tttactcggc atccagcctc1801 tactctggag tcccttctcg cttctctggt agccgttccg ggacggattt cactctgacc1861 atcagcagtc tgcagccgga agacttcgca acttattact gtcagcaatc ttcttattct1921 ctgatcacgt tcggacaggg taccaaggtg gagatcaaag gtactactgc cgctagtggt1981 agtagtggtg gcagtagcag tggtgccgag gttcagctgg tggagtctgg cggtggcctg2041 gtgcagccag ggggctcact ccgtttgtcc tgtgcagctt ctggcttcaa cttttcttct2101 tcttctatac actgggtgcg tcaggccccg ggtaagggcc tggaatgggt tgcatctatt2161 tcttcttctt atggctatac ttattatgcc gatagcgtca agggccgttt cactataagc2221 gcagacacat ccaaaaacac agcctaccta caaatgaaca gcttaagagc tgaggacact2281 gccgtctatt attgtgctcg cactgttcgt ggatccaaaa aaccgtactt ctctggttgg2341 gctatggact actggggtca aggaaccctg gtcaccgtct cctcggccga caaaactcac2401 acatgcggcc ggccctctgg ttccggtgat tttgattatg aaaagatggc aaacgctaat2461 aagggggcta tgaccgaaaa tgccgatgaa aacgcgctac agtctgacgc taaaggcaaa2521 cttgattctg tcgctactga ttacggtgct gctatcgatg gtttcattgg tgacgtttcc2581 ggccttgcta atggtaatgg tgctactggt gattttgctg gctctaattc ccaaatggct2641 caagtcggtg acggtgataa ttcaccttta atgaataatt tccgtcaata tttaccttcc2701 ctccctcaat cggttgaatg tcgccctttt gtctttagcg ctggtaaacc atatgaattt2761 tctattgatt gtgacaaaat aaacttattc cgtggtgtct ttgcgtttct tttatatgtt2821 gccaccttta tgtatgtatt ttctacgttt gctaacatac tgcgtaataa ggagtcttaa2881 tcatgccagt tcttttggct agcgccgccc tataccttgt ctgcctcccc gcgttgcgtc2941 gcggtgcatg gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg3001 attcaccact ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac3061 caacccttgg cagaacatat ccatcgcgtc cgccatctcc agcagccgca cgcggcgcat3121 ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac3181 ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg3241 aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt3301 cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt3361 ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac3421 gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag3481 ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt3541 gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatt cccccttaca3601 cggaggcatc aagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga3661 agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac3721 atctgtgaat cgcttcacga ccacgctgat gagctttacc gcaggatccg gaaattgtaa3781 acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc3841 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga3901 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag3961 ggcgaaaaac cgtctatcag ggctatggcc cactacgtga accatcaccc taatcaagtt4021 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta4081 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag4141 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg4201 cgcttaatgc gccgctacag ggcgcgtccg gatcctgcct cgcgcgtttc ggtgatgacg4261 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg4321 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag4381 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga4441 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag4501 aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt4561 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc4621 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa4681 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa4741 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc4801 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc4861 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag4921 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga4981 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc5041 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac5101 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg5161 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca5221 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa5281 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa5341 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt5401 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag5461 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat5521 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc5581 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa5641 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca5701 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa5761 cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt5821 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc5881 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact5941 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc6001 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg6061 ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct6121 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc6181 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag6241 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac6301 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg6361 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt6421 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac6481 attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaa (SEQ ID NO: 249)

Library F is an Fab-phage library that was constructed by introducingdegenerate codons into positions in CDR-H1, CDR-H2, CDR-H3 and CDR-L3 ofa single human Fab framework. The loop length of the CDR-L3 and/orCDR-H3 in Library F can vary as shown in the table below. The libraryhas a total diversity of 3×10¹⁰ unique clones, and the details of thelibrary design are shown in Table 2 below, where the bolding in theCDR-L3 and CDR-H3 regions represents positions that were replaced byrandom loops of all possible varying lengths, as indicated.

TABLE 2 CDR Sequences of Library F clones CDR-L3 (SEQ ID NO: 262) LoopLength (8-12 aa) Q Q Z Z Z Z PL IL T Z = 25% Y, 20% S, 20% G, 10% A and5% each of F, W, H, P, V CDR-H1 (SEQ ID NO: 263) Position 29 30 31 32 3334 IL YS YS YS YS IM CDR-H2 (SEQ ID NO: 264) Position 50 51 52 52a 53 5455 56 57 58 YS I YS PS YS YS GS YS T YS CDR-H3 (SEQ ID NO: 265) LoopLength (5-22 aa) Position 94 95 96 97 98 99 100 100a 100b 100c 101 R Z ZZ Z Z Z Z AG FILM D Z = 25% Y, 20% S, 20% G, 10% A, and 5% each of F, W,H, P, V

The nucleotide sequence of the vector encoding Library F is shown below:

FEATURES Location/Qualifiers promoter 536 . . . 752 /note = “Pho A” ORFcomplement(4052 . . . 4913) /note = “AmpR” ORF 2461 . . . 2925/note = “III gene (G2-CT)” sig_peptide 804 . . . 872/note = “ST2 secr signal” sig_peptide 1669 . . . 1737/note = “ST2 secr signal” misc_feature 1747 . . . 2124 /note = “VH”misc_feature 1197 . . . 1526 /note = “CL” misc_feature 876 . . . 1196/note = “VL” misc_feature 2125 . . . 2433 /note = “CH1” misc_feature2041 . . . 2085 /note = “CDRH3” misc_feature 1894 . . . 1923/note = “CDRH2” misc_feature 1527 . . . 1550 /note = “FLAG tag”misc_feature 1023 . . . 1043 /note = “CDRL2” misc_feature1146 . . . 1163 /note = “CDRL3” misc_feature 1831 . . . 1848/note = “CDRH1” misc_feature 2434 . . . 2448 /note = “Hinge”misc_feature 957 . . . 971 /note = “CDRL1” misc_feature 2449 . . . 2460/note = “dimerization domain”   1 ggaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc  61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg cgccattcgc cattcaggct 481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcg catgcgacca 541 acagcggttg attgatcagg tagagggggc gctgtacgag gtaaagcccg atgccagcat 601 tcctgacgac gatacggagc tgctgcgcga ttacgtaaag aagttattga agcatcctcg 661 tcagtaaaaa gttaatcttt tcaacagctg tcataaagtt gtcacggccg agacttatag 721 tcgctttgtt tttatttttt aatgtatttg taactagtac gcaagttcac gtaaaaaggg 781 tatgtagagg ttgaggtgat tttatgaaaa agaatatcgc atttcttctt gcatctatgt 841 tcgttttttc tattgctaca aatgcctatg catccgatat ccagatgacc cagtccccga 901 gctccctgtc cgcctctgtg ggcgataggg tcaccatcac ctgccgtgcc agtcagtccg 961 tgtccagcgc tgtagcctgg tatcaacaga aaccaggaaa agctccgaag cttctgattt1021 actcggcatc cagcctctac tctggagtcc cttctcgctt ctctggtagc cgttccggga1081 cggatttcac tctgaccatc agcagtctgc agccggaaga cttcgcaact tattactgtc1141 agcaatcttc ttattctctg atcacgttcg gacagggtac caaggtggag atcaaacgaa1201 ctgtggctgc accatctgtc ttcatcttcc cgccatctga ttcacagttg aaatctggaa1261 ctgcctctgt tgtgtgcctg ctgaataact tctatcccag agaggccaaa gtacagtgga1321 aggtggataa cgccctccaa tcgggtaact cccaggagag tgtcacagag caggacagca1381 aggacagcac ctacagcctc agcagcaccc tgacgctgag caaagcagac tacgaaaaac1441 ataaagtcta cgcctgcgaa gtcacccatc agggcctgag ctcgcccgtc acaaagagct1501 tcaacagggg agagtgtggt ggttctgatt acaaagatga cgatgacaaa taattaactc1561 gaggctgagc aaagcagact actaataaca taaagtctac gccggacgca tcgtggccct1621 agtacgcaag ttcacgtaaa aagggtaact agaggttgag gtgattttat gaaaaagaat1681 atcgcatttc ttcttgcatc tatgttcgtt ttttctattg ctacaaacgc gtacgctgag1741 atctccgagg ttcagctggt ggagtctggc ggtggcctgg tgcagccagg gggctcactc1801 cgtttgtcct gtgcagcttc tggcttcaac ttttcttctt cttctataca ctgggtgcgt1861 caggccccgg gtaagggcct ggaatgggtt gcatctattt cttcttctta tggctatact1921 tattatgccg atagcgtcaa gggccgtttc actataagcg cagacacatc caaaaacaca1981 gcctacctac aaatgaacag cttaagagct gaggacactg ccgtctatta ttgtgctcgc2041 actgttcgtg gatccaaaaa accgtacttc tctggttggg ctatggacta ctggggtcaa2101 ggaaccctgg tcaccgtctc ctcggcctcc accaagggtc catcggtctt ccccctggca2161 ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt caaggactac2221 ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc2281 ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc2341 tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc cagcaacacc2401 aaggtcgaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg cggccggccc2461 tctggttccg gtgattttga ttatgaaaag atggcaaacg ctaataaggg ggctatgacc2521 gaaaatgccg atgaaaacgc gctacagtct gacgctaaag gcaaacttga ttctgtcgct2581 actgattacg gtgctgctat cgatggtttc attggtgacg tttccggcct tgctaatggt2641 aatggtgcta ctggtgattt tgctggctct aattcccaaa tggctcaagt cggtgacggt2701 gataattcac ctttaatgaa taatttccgt caatatttac cttccctccc tcaatcggtt2761 gaatgtcgcc cttttgtctt tagcgctggt aaaccatatg aattttctat tgattgtgac2821 aaaataaact tattccgtgg tgtctttgcg tttcttttat atgttgccac ctttatgtat2881 gtattttcta cgtttgctaa catactgcgt aataaggagt cttaaagctc caattcgccc2941 tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac3001 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctgcattaat3061 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc3121 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg3181 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag3241 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc3301 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag3361 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga3421 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc3481 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg3541 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt3601 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca3661 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca3721 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag3781 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca3841 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg3901 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa3961 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta4021 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag4081 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga4141 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac4201 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc4261 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta4321 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac4381 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat4441 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa4501 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg4561 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag4621 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc4681 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct4741 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat4801 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg4861 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc4921 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta4981 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctg(SEQ ID NO: 248)

In a further aspect, there is provided an antibody or antibody fragmentcomprising any one of CDR regions outlined in FIG. 2 , FIG. 5 or FIG. 9. For antibodies or antigen-binding fragments thereof shown in FIG. 2 orderived from those shown in FIG. 2 , the antibody or fragment contains aCDR-L1 that includes the amino acid sequence SVSSA (SEQ ID NO: 240), aCDR-L2 that includes the amino acid sequence SASSLYS (SEQ ID NO: 241),and one of the combinations of CDR-L3, CDR-H1, CDR-H2 and CDR-H3 shownin FIG. 2 .

For antibodies or antigen-binding fragments thereof shown in FIG. 5 orderived from those shown in FIG. 5 , the antibody or fragment contains aCDR-L1 that includes the amino acid sequence SVSSA (SEQ ID NO: 240), aCDR-L2 that includes the amino acid sequence SASSLYS (SEQ ID NO: 241), aCDR-L3 that includes the amino acid sequence X₁-X₂-X₃-X₄-X₅—X₆ (SEQ IDNO: 242), where X₁, X₂, X₃, and X₄ are Y, S, G, A, F, W, H, P or V andX₅ is P or L and X₆ is I or L; a CDR-H1 that includes the amino acidsequence X₁-X₂-X₃-X₄-X₅-X₆ (SEQ ID NO: 243), where X₁ is I or L, X₂, X₃,X₄, and X₅ are Y or S and where X₆ is I or M; and a CDR-H2 that includesthe amino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉ (SEQ ID NO: 244),where X₁, X₂, X₄, X₅, X₆, X₈, and X₉ is Y or S, X₃ is P or S, and whereX₇ is G or S; and one of the CDR-H3 sequences shown in FIG. 5 .

For antibodies or antigen-binding fragments thereof shown in FIG. 9 orderived from those shown in FIG. 9 and were identified from Library F,the antibody or fragment contains a CDR-L1 that includes the amino acidsequence SVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acidsequence SASSLYS (SEQ ID NO: 241), a CDR-L3 that includes the amino acidsequence X₁-X₂-X₃-X₄-X₅-X₅ (SEQ ID NO: 242), where X₁, X₂, X₃, and X₄are Y, S, G, A, F, W, H, P or V and X₅ is P or L and X₆ is I or L; aCDR-H1 that includes the amino acid sequence X₁-X₂-X₃-X₄-X₅-X₆ (SEQ IDNO: 243), where X₁ is I or L, X₂, X₃, X₄, and X₅ are Y or S and where X₆is I or M; and a CDR-H2 that includes the amino acid sequenceX₁-I-X₂-X₃-X₄-X₅—X₆-X₇-X₈-T-X₉ (SEQ ID NO: 244), where X₁, X₂, X₄, X₅,X₆, X₈, and X₉ is Y or S, X₃ is P or S, and where X₇ is G or S; and oneof the combinations of CDR-L3 and CDR-H3 sequences shown in FIG. 9(where the Library column indicates F).

For antibodies or antigen-binding fragments thereof shown in FIG. 9 orderived from those shown in FIG. 9 and were identified from Library G,the antibody or fragment contains a CDR-L1 that includes the amino acidsequence Q-X₁-X₂-X₃-X₄-X₅ (SEQ ID NO: 245), where X₁, X₂, X₃, X₄, and X₅are Y, S or, G; a CDR-L2 that includes the amino acid sequenceX₁-A-S—X₂-L-Y (SEQ ID NO: 246), where X₁ and X₂ are Y, S or, G; a CDR-H1that includes the amino acid sequence X₁-X₂-X₃-X₄-X₅-X₆ (SEQ ID NO:247), where X₁ is I or L, X₂, X₃, X₄, and X₅ are Y, S or G and where X₆is I or M; a CDR-H2 that includes the amino acid sequenceX₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉ (SEQ ID NO: 244), where X₁, X₂, X₄, X₅,X₆, X₈, and X₉ is Y or S, X₃ is P or S, and where X₇ is G or S; and oneof the combinations of CDR-L3 and CDR-H3 sequences shown in FIG. 9(where the Library column indicates G).

Preferably, the antibody or antibody fragment is selected from the groupconsisting of antibodies or antibody fragments comprising CDRL3, CDRH1,CDRH2 and CDRH3 of any one of clones WY574B, WY574E, WY574F, WY677C andWY677D described herein, the CDRH3 regions shown in FIG. 5 or thecombinations of CDRL3 and CDRH3 regions shown in FIG. 9 . In oneembodiment, the antibody or antibody fragment is for the treatment ofcancer, e.g., Her-2 positive cancer, preferably selected from the groupconsisting of breast cancer, ovarian cancer, uterine cancer and stomachcancer.

In a further aspect, there is provided a method of treating a disorderthat is associated with aberrant expression and/or activity of thecell-surface target against which the antibody has been selectedcomprising administering to the patient a therapeutically effectiveamount of the antibody or antibody fragment described herein.

In a further aspect, there is provided a method of treating a cancer,such as a Her-2 positive cancer, preferably selected from the groupconsisting of breast cancer, ovarian cancer, uterine cancer and stomachcancer, in a patient comprising administering to the patient atherapeutically effective amount of the antibody or antibody fragmentdescribed herein.

In a further aspect, there is provided a use of the antibody or antibodyfragment described herein for the treatment of a cancer, such as a Her-2positive cancer, preferably selected from the group consisting of breastcancer, ovarian cancer, uterine cancer and stomach cancer.

In a further aspect, there is provided a use of the antibody or antibodyfragment described herein in the preparation of a medicament for thetreatment of a cancer, such as Her-2 positive cancer, preferablyselected from the group consisting of breast cancer, ovarian cancer,uterine cancer and stomach cancer.

The following examples are illustrative of various aspects of theinvention, and do not limit the broad aspects of the invention asdisclosed herein.

EXAMPLES Example 1. Rapid Isolation of Antibody Fragments toCell-Surface Targets

The considerable heterogeneity of cell-surfaces makes selection ofphage-displayed antibody libraries against cell-surface antigenschallenging. We report the development of a unique methodology forrapidly isolating phage-displayed antibody fragments to cell-surfacetargets, using the oncogenic human epidermal growth factor receptor 2(Her2) as a model. Synthetic phage-displayed libraries were selected inparallel on Her2-positive and negative cells. Following three rounds ofselection, the output phage pools were analyzed by Illumina deepsequencing. Comparisons of the sequences from the positive and negativeselection pool allowed sequences specific to the antigen-expressingcell-line to be readily identified from background phage clones. A PCRamplification strategy that used primers specific to the unique heavychain third hypervariable loop enabled the recovery of clones from thepositive selection pool, which represented 2.95% to 0.25% of the phagepool. Binding kinetics measured by surface plasmon resonance showed thatall of the recovered antibody fragments bind to Her2 specifically andwith high affinity. Three of the isolated antibody fragments wereassayed for specific binding to Her2 expressed on the surface oftransiently transfected cells and a Her2+ breast cancer cell-line byflow cytometry and immunofluorescence. These antibody fragmentsdisplayed specific binding to cell-surface Her2, demonstrating that ourmethodology, termed CellectSeq, is amenable to the rapid identificationof high affinity antibody fragments specific to cell-surface epitopes.Together, these results suggest that the CellectSeq approach canincrease the efficiency of library selections to cell-surface targetsand eliminates the need for purified antigen.

Materials and Methods

Cell Culture

293T cells were cultured in Dulbecco's Modified Eagle medium (DMEM)supplemented with 10% heat inactivated fetal bovine serum (FBS). Humanbreast cancer cell lines T47D and BT474 cells were cultured in DMEMsupplemented with 10% FBS and penicillin and streptomycin. All cellswere cultured at 37° C. in a humid incubator with 5% CO₂.

Phage-Displayed Fab Library and Screening

Selections were performed using Library F, a single framework human Fablibrary constructed similarly to previously described libraries [28,29].Briefly, a phagemid vector was engineered to bivalent display a humanFab on the pIII protein of M13 bacteriophage. All three heavy chain CDRsand the light chain CDR3 were mutagenized using Kunkel mutagenesis andtailored oligonucleotide mixtures. Solvent assessable residues of CDRsH1 and H2 were restricted to tyrosine and serine residues, whereas CDRsH3 and L3 were allowed a much more complex chemical diversity of thefollowing composition: 25% Tyr, 20% Ser, 20% Gly, 10% Ala, and 5% eachof Phe, Trp, His, Pro and Val. The CDR H3 and L3 lengths were variedbetween 5 to 22 and 8 to 12 residues, respectively.

Library F was cycled through three rounds of selection, each consistingof a pre-absorption step followed by a positive selection step. For thepre-absorption step, 293T cells were trypsinized briefly andre-suspended in a single cell suspension in DMEM with 10% FBS. Tenmillion cells were pelleted at 1200 rpm for three minutes and cells weremixed with approximately 10¹² cfu of library F phage in DMEM containing10% FBS, 50 mM HEPES, 2 mM EDTA. The cells and library were incubatedfor 1.5 to 2 hours at 4° C. with gentle rocking, after which the cellswere pelleted and the library supernatant was used in the followingpositive selection step.

For positive selection, 293T cells were harvested and plated at 2×10⁶cells in 150 mm tissue culture dishes (BD Falcon). Twenty-four hoursafter plating, cells were co-transfected with a Her2 expression plasmid(8 μg) and a GFP expression plasmid (2 μg) using Fugene 6 (Roche AppliedSciences), following the manufacturer's instructions. Approximately 48hours post-transfection, cells were harvested as described above for thepre-absorption step. Five million cells were pelleted and re-suspendedin the phage library supernatant from the pre-absorption step. Thelibrary and transfected cells were incubated for 2 hours at 4° C. withgentle shaking. Following incubation, cells were pelleted as before, thesupernatant was discarded, and cells were re-suspended in coldphosphate-buffered saline (PBS). This process was repeated for a totalof two washes for round one and three washes for rounds two and three.To obtain the negative selection pool for Illumina sequencing, Library Fwas also selected for three rounds against 5 million untransfected 293Tcells, using the same methods described for the positive selection step.

Positively selected phage were amplified similarly to previous describedmethods [30]. Briefly, XL1blue cells were grown to an OD₆₀₀ of 0.8 in2YT media containing 10 μg/ml tetracycline. Following washing of thepositively selected cells, 3 ml of the XL1blue culture was addeddirectly to the cell pellet. Cells and bacteria were incubated for 30 to40 minutes at 37° C. with gentle shaking and approximately 10¹⁰ cfu ofM13 K07 helper phage was added. The culture was incubated for 45 minutesat 37° C., shaking at 200 rpm, and then transferred to a 40 ml 2YTculture (100 μg/ml carbenicillin, 25 μg/ml kanamycin). The culture wasgrown overnight at 37° C., shaking at 200 rpm. The amplified phageculture was harvested for subsequent selection rounds as previouslydescribed [30].

Illumina Sequencing and PCR Amplification of Phagemid Clones

The round three positive and negative selection pool phage, along withthe naïve library, were infected into XL1blue cells and grown overnightin 2YT supplement with 100 μg/ml carbenicillin. Cultures wereminiprepped (Qiagen) to obtain phagemid DNA to use as the templates fora PCR with individual forward primers comprised of an adaptor sequence(5′AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′) (SEQID NO: 1), a five base pair barcode sequence (positive pool: 5′-GAGTA-′3(SEQ ID NO: 2); negative pool: 5′-CCAAA-′3 (SEQ ID NO: 3); naïvelibrary: 5′-TTGTT-3′ (SEQ ID NO: 4)) and an annealing site to the thirdantibody framework region of the heavy chain (5′-GTCTATTATTGTGCTCGC-3′)(SEQ ID NO: 5). For all phage pools, a reverse primer containing asecond Illumina-compatible adaptor region(5′-CAAGCAGAAGACGGCATACGAGCTCTTC-3′) (SEQ ID NO: 6) and an annealingsite to the phagemid vector (5′-TCCTTGACCCCAGTAGTC-3′) (SEQ ID NO: 7)was used. PCR reactions were performed with the high fidelity polymerasePhusion (Finnzyme) and 400 to 600 ng of template DNA. Reactions weresubjected to 15 cycles of annealing and extension, consisting of 30s at57° C. and 45s at 72° C. PCR products were digested with ExoI (USB), SAP(USB), and Dpn1 (NEB) and then purified on a PCR purification column(Qiagen). Successful amplification of the correct DNA fragment from eachphage pool was verified by agarose gel electrophoresis. The amplifiedDNA fragments were pooled and subjected to Illumina DNA sequencing on anIllumina GAII, with 72 base pair reads. Each sequencing read wasassigned to its correct pool on the basis of its unique barcodesequence. The reads were filtered according to their Phred score [31].Since a constant aligner region was sequenced, these regions were usedto optimize the phred score cutoffs. Briefly, all sequences with phredscores of 20 and higher for every base were kept. A tolerance number (5)of medium quality (phred score higher than 15) was allowed. DNAsequences were translated to decode the sequence of the heavy chainCDR3.

To rescue individual clones from the positive selection pool, primers(described below) were phosphorylated as previously described [30]. Thephosphorylated primers were then used in a PCR reaction, in which phagepool DNA was used as a template. The amount of DNA template per reactionwas varied between 1 to 100 ng. The amount of DNA template varied withthe prevalence of the given clone in the IIlumina pool, with greatestamount of DNA template (100 ng) being used in PCR reactions to rescuethe least prevalent clones. Reactions were performed with the highfidelity polymerase Phusion (Finnzyme), using the manufacturerrecommended conditions. Reactions were subjected to 30 to 35 cycles ofannealing and extension, consisting of 30s at 65 or 68° C. and 180s at72° C. PCR products were confirmed by agarose gel electrophoresis andapproximately 50 ng of the PCR product was used directly in ligationreaction (400 U T4 ligase, NEB). Ligations were incubated overnight atroom temperature, and then heat inactivated at 65° C. for 10 minutes.Following the PCR, Dpn1 (NEB) was added to digest template DNA presentin the reactions and samples were transformed into chemically competentXL1blue cells. Rescued transformations were plated on 2YT agar plateswith carbenicillin and incubated overnight at 37° C. Single colonieswere inoculated into 96-well culture plates for overnight growth ofsingle phage clones as previously described [30]. The heavy and lightchains of individual phage clones were PCR amplified and the PCRproducts were sequenced to ensure the recovery of clones with thedesired CDR H3.

Vectors and Primers

For PCR recovery the following phosphorylated forward and reverseprimers were used to recover the phage-Fab clones:

WY574B: 5′-CCAGTAATGAACAACAGC-3′, (SEQ ID NO: 250)5′-TACGGTTACGTTTCTGGT-3′; (SEQ ID NO: 8) WY574E:5′-AGCCGGAACCCAACCGCG-3′, (SEQ ID NO: 251) 5′-TACCCGTCTTACGGTTTG-3′;(SEQ ID NO: 9) WY574F: 5′-AGCGTAAACAGAAGAACCCCA-3′, (SEQ ID NO: 252)5′-TGGTCTCCGGCTTCTTGGTCT-3′; (SEQ ID NO: 10) WY677C:5′-ACCCCACCAGTAGTAAGA-3′, (SEQ ID NO: 253) 5′-CCGTGGTCTGGTTACTCT-3′;(SEQ ID NO: 11) WY677D: 5′-GTACGGAATGTACGGATGCGG-3′, (SEQ ID NO: 254)5′-TACTCTTACTGGGGTCCGTACTAC-3′. (SEQ ID NO: 12)

Heavy (V_(H)) and light-chain (V_(L)) variable regions were amplifiedfor sequencing with the following primers that add M13 forward andreverse binding sites, respectively:

V_(H): (SEQ ID NO: 13) 5′-TGTAAAACGACGGCCAGTGGACGCATCGTGGCCCTA-3′,(SEQ ID NO: 14) 5′-CAGGAAACAGCTATGACCCCTTGGTGGAGGCCGAG-3′; VL:(SEQ ID NO: 15) 5′-TGTAAAACGACGGCCAGTCTGTCATAAAGTTGTCACGG-3′,(SEQ ID NO: 16) 5′-CAGGAAACAGCTATGACCCCTTGGTACCCTGTCCG-3′

Her2 and EGFR were both expressed from pCDNA3 (Invitrogen) [32,33], andGFP was expressed from a previously reported plasmid [34].

Protein Expression and Purification

Fab proteins were expressed in 55244 E. coli from the phage displayphagemid engineered with an amber stop codon between the Fab and pIIIproteins, introduced by a standard Kunkel mutagenesis reaction [30].Single colonies of each clone were grown overnight at 30° C. in 2YTmedia supplemented with 50 μg/ml carbenicillin and 25 μg/ml kanamycin.Overnight cultures were centrifuged at 3000 g for 10 minutes and pelletswere re-suspended in 25 ml of complete CRAP media [30]. Ten millilitersof the re-suspended culture was used to inoculate 1 L of CRAP media,which was subsequently grown for 24 to 27 hours at 30° C., pelleted,re-suspended in 25 ml of PBS, and frozen. After thawing, 15 mg oflysozyme (Bioshop) and 30 μl of DNase I (deoxyribonuclease I, Fermentus)was added to 30 ml of cell suspension and cells were lysed bysonication. Following centrifugation to pellet cell debris, Fabsupernatants were loaded onto fast-flow rProtein A-Sepharose (GEHealthcare) pre-equilibrated in PBS. Columns were washed with PBS,eluted with 50 mM NaH₂PO₄, 100 mM H₃PO₄, 140 mM NaCl, pH 2.5. Eluateswere neutralized with 1 M Na₂HPO₄, 140 mM NaCl. Recovered Fab proteinswere analyzed by SDS-PAGE and quantified using a Bradford assay(Bio-Rad).

Surface Plasmon Resonance

The binding affinities and kinetic parameters for interactions betweenHer2 specific Fabs and recombinant Her2 (R&D Systems) were measured bysurface plasmon resonance using a ProteOn XPR36 instrument (Bio-Rad).HER2 was immobilized on a GLC chip by standard amine coupling chemistryand serial dilutions of Fab in PBS with 0.05% Tween 20 were injectedover the Her2 and blank channels (for reference subtraction) for 60seconds at a flow rate of 100 μl/min, followed by ten minutes of bufferto monitor Fab dissociation. The chip surface was regenerated with 0.85%H₃PO₄ prior to new analyte injection. Kinetic parameters were determinedby globally fitting a reference cell-subtracted concentration series toa 1:1 (Langmuir) binding model.

Flow Cytometry and Immunofluorescence Staining

For flow cytometric analysis of transfected 293T cells, 3×10⁶ cells wereplated on 10 cm dishes (BD Falcon). Twenty-four hours after plating,cells were transfected with 10 μg of a Her2, EGFR, or GFP expressionvector using Fugene 6 (Roche Applied Sciences), following themanufacturer's instructions. Approximately 24 hours post-transfection,cells were harvested using a cell scraper into PBS containing 2% FBS(wash buffer). The cells were washed once with wash buffer andre-suspended into a single cell suspension. Approximately 1.0 to 1.5×10⁶cells were placed into 1.5 ml tubes for staining with individual Fabclones. First, cells were incubated for 45 minutes at room temperaturein PBS containing 2% FBS to block non-specific epitopes. Next, cellswere incubated with 2 μg of the Her2 specific Fabs (diluted in washbuffer) for 20 minutes at room temperature and then washed twice withwash buffer. The samples were incubated for 20 minutes at roomtemperature with a 1:100 dilution of anti-FLAG-Alexa488 secondaryantibody (Cell Signaling) in wash buffer. Finally, cells were washedtwice and re-suspended in 0.5 ml of PBS for analysis on a BD FacsAria Iflow cytometer (BD Biosciences).

For flow cytometric analysis of Fab binding to the human cancercell-lines, 5×10⁵ BT474 and T47D cells were plated per well of 6-wellplates (BD Falcon). Approximately 48 hours after plating, media wasaspirated from the 6-well plates and cells were washed twice with coldPBS. Wells were then blocked with wash buffer for 45 minutes at 4° C.The blocking solution was aspirated and 4 μg of the Fab sample in 0.5 mlof wash buffer was added to the appropriate well. Wells were washedtwice with wash buffer, and then incubated with secondary antibody asabove for 30 minutes at 4° C. Wells were washed three times; cells wereharvested into PBS using a cell scraper, and analyzed as above.

Immunofluorescence for cell-surface HER2 was carried out on intact cellsseeded on round glass coverslips uncoated or coated with 50 μg/mLpoly-D-lysine (BD Biosciences). 48 hours post-seeding orpost-transfection with a plasmid encoding HER2, the cells were washedwith ice-cold PBS containing 1 mM MgCl₂ and 1 mM CaCl₂ on ice. Thesubsequent steps were performed at 4° C., unless otherwise indicated.The cells were fixed for 10 min with 3% paraformaldehyde (ElectronMicroscopy Sciences) and then stained with anti-HER2 Fab protein (5mg/ml) in 1% (wt/vol) BSA for 1 h followed by extensive washing andincubation with Alexa488-conjugated secondary antibody against aFlag-epitope on the C terminus of the Fab light-chain. The nuclei werestained using the Hoechst dye (Invitrogen) and then mounted with ProLongantifade reagent (Invitrogen). The images were acquired using the WaveFXspinning disk confocal microscope by Quorom Technologies Inc.Acquisition parameters were adjusted to exclude saturation of thepixels. For assessing binding specificity in HER2+ (BT474) and HER2−(T47D) cells, such parameters were kept constant between the two celllines.

Results and Discussion

Library Screening Against Her2-Transfected Cells and Illumina SequenceAnalysis

We subjected the synthetic Fab library F to three rounds of selection on293T cells transiently transfected to express Her2 (FIG. 1 a ). To helpreduce background from phage binding to undesired cell-surface epitopesand non-specific binding phage clones, the library was incubated withuntransfected 293T cells prior to incubation with the Her2 expressingcells. These undesired background phage were removed with the cellpellet and the library phage left in solution were incubated with theHer2 transfected cells. After washing away non-binding phage, theremaining phage, which should include the Her2 specific binders(positive selection pool), were amplified in E. coli. We also carriedout three rounds of selection against untransfected 293T cells. Thisnegative selection was carried out with the rational that the sequencesobtained from this pool represent undesired background clones that areunlikely to be Her2 specific binders. As such, comparing the sequencesfrom the positive and negative selections should help readily identifysequences in the positive pool that arise from phage clones binding toundesired epitopes.

The positive and negative selection pools, and the naïve library, werenext subjected to Illumina sequencing analysis. Of the 100 mostfrequently observed CDR H3 sequences in the positive pool, whichrepresent anywhere from 0.06 to 14.94% of the total number of sequencesobtained, 20 were also present in the negative selection pool (FIG. 5 ).A similar number of sequences, 20, from the positive selection pool alsooverlap with the naïve library pool. Sixteen of the sequences observedin the naïve library are also present in the negative selection pool. Asexpected, sequences in the naïve library exhibited a much greater degreeof diversity than the sequences of the two selected pools.

PCR Recovery of Her2 Specific Clones from the Positive Selection Pool

Single clones of interest, identified from the Illumina sequencingresults, were isolated from the positive selection output pool using aPCR based recovery method in which phosphorylated primers annealed tounique CDR H3 sequences (FIG. 1 b ). The primers were designed so thatthe 5′ ends of the forward and reverse primers were abutting, resultingin the amplification of the complete phagemid clone vector. Following ablunt-end ligation and transformation into E. coli, single colonies canbe isolated and sequenced to verify recovery of the desired CDR H3.Using this method, we successfully recovered five unique phage clonesfrom the positive selection pool (FIG. 2 ). The successfully recoveredclones vary in their abundance in the positive pool, with the leastabundant clone representing only 0.25% of the pool. Of note, five of thePCR reactions we attempted failed to generate a PCR product. This may bedue to their low abundance in the output pool used for the PCR template,as each of these five clones represented less than 0.5% of the pool.

Binding Kinetics of Recovered Anti-Her2 Clones

Kinetic analysis of the purified Fabs by SPR shows that the fiverecovered Fab clones bind to recombinant Her2 with high affinities(Table 3), with K_(D) values ranging from 4 nM to 75 nM. These datasuggest that the cell-surface selection methodology presented here canbe used to rapidly recover multiple Fab clones that bind with highaffinity to the target of interest. We chose three of the five Fabclones, WY547B, WY574E, and WY574F, for further analysis based on theobservation that they exhibit a range of affinities encompassing thehighest (WY574F), lowest (WY574E), and an intermediate (WY574B) affinityvalue.

TABLE 3 Binding Kinetics of anti-Her2 Fab clones Fab K_(a) (M⁻¹s⁻¹)K_(d) (s⁻¹) K_(D) (nM) WY574B  1.0 × 10⁵  1.4 × 10⁻³ 14 WY574E  1.9 ×10⁵  6.8 × 10⁻⁴ 4 WY574F  9.7 × 10³  7.3 × 10⁻⁴ 75 WY6770 4.37 × 10⁴1.62 × 10⁻³ 37 WY677D 4.01 × 10⁵ 1.08 × 10⁻² 27Cell-Surface Specificity of Anti-Her2 Fabs

Next, the specificity of Fab clones WY574B, WY574E, and WY574E, wasexamined by flow-cytometry using 293T cells transiently transfected withHer2 or EGFR, which is also a member of the EGFR receptor family. Afluorescence shift was observed in the Her2-transfected cell populationfor all three Fab clones (FIG. 3 a ). A similar shift in fluorescencestaining was not observed in the EGFR-transfected cell population or theunstained Her2-transfected 293T cell population. This data suggests thatthe recovered Fabs are binding specifically to the Her2 transfected cellpopulation. We also evaluated the specificity of the Fab clones byflow-cytometric analysis with the Her2 positive breast cancer cell-lineBT474 and the Her2 negative breast cancer cell-line T47D (FIG. 3 b ). Asexpected, a drastic shift in the fluorescence signal of the BT474 cellpopulation was observed in the presence of the anti-Her2 Fabs. Incontrast, little or no binding of the three Fabs was detected in theHer2 negative T47D cell population.

Finally, we sought to confirm the specificity of the three Fabs for Her2presented on the cell-surface by immunofluorescent staining (IF) ofHer2-transfected 293T cells and a Her2 expressing cancer cell-line.Fluorescent staining of each Fab clone was observed around the cellperiphery in the Her2-transfected 293T cells (FIG. 4 a ). Consistentwith staining pattern observed in the Her2-transfected cells,fluorescent staining of the Her2 positive BT474 cancer-cell line wasalso evident by IF (FIG. 4 b ). In contrast, no specific staining wasobserved for the Her2-negative T47D cancer cell-line. Collectively,these data strongly demonstrate that the recovered Fab clones bindspecifically to Her2 presented in the context of the cell-surface.

Selection of phage-displayed antibody libraries against cell-surfaceantigens is often challenging, as the vast array of epitopes presentedon the cell-surface gives rise to a high degree of background bindingand poor enrichment of clones specific to the target of interest. Aunique aspect of the methodology described here is the use of deepsequencing to identify phage clones specific to the cell-surface antigenof interest. Here, sequences distinctive/exclusive to the positiveselection output pool represent clones that have a high probability ofbeing specific for the target antigen. In addition, combiningcell-surface selections with deep sequencing allows rare clones to beidentified. It is unlikely that the degree of clonal diversity weobserve by deep sequencing could be resolved using traditional phagedisplay methodologies, in part because of the practical limitations ofmanually screening sufficient numbers of single phage clones to retrievea similar level of sequence diversity. Although a variety of factors caninfluence clonal diversity during the selection process, such as thegrowth advantage of certain clones, selection methodologies also tend topreferentially enrich for higher affinity binders. As a consequence, thediversity of the sequences recovered in later rounds may be diminished.However, high throughput DNA sequencing is becoming an increasinglyaccessible technology, as evidenced by recent reports that made use ofdeep sequencing approaches to characterize human antibody libraries andV-gene repertoires of immunized mice [25,26].

We reasoned that Her2 would be an ideal model given the existence of awell-characterized therapeutic monoclonal antibody specific for Her2,which is reflective of our goal of applying the CellectSeq methodologyto isolating stable, high affinity, antibody fragments specific totherapeutically relevant cell-surface proteins. Trastuzumab (Genentech,also known as Herceptin) is a humanized IgG1 specific for theextracellular domain of Her2 [22], which is approved for clinicaltreatment of Her2 positive breast cancer. Although Trastuzumabrepresents a very successful therapeutic option for patients, not allHer2 positive cancers are responsive to Trastuzumab treatment [23]. Inaddition, resistance to Trastuzumab may also develop during the courseof treatment [20,24]. The synthetic antibody fragments we haveidentified using the CellectSeq method exhibit binding characteristicsthat are highly desirable for potential new therapeutic antibodycandidates. Specifically, the synthetic antibody fragments we haveisolated bind with both high affinity and specificity to Her2.

The five synthetic antibody fragments rescued from our positiveselection pool exhibit specific binding to Her2, both by SPR analysis torecombinant Her2 and by flow-cytometry and IF to cell-surface Her2.However, it is also important to note that the methodology we reporthere may allow for the identification of antibody fragments specific forproteins that are over-expressed as a consequence of the over-expressionof Her2 itself.

Of the ten unique CDR H3 clones we attempted to rescue, five failed togenerate a PCR product. This may be due to factors that include thesequence and length of the CDR H3, the abundance of the template in thepositive selection pool, or the design of the PCR primers. It isimportant to consider, however, that these factors were not optimized inthis study. In light of this observation, the number of phagemid cloneswe successfully rescued is considerably high. Another importantconsideration is whether the methodology presented here introduces biasinto the final sequence analysis. For example, many of the sequences inthe naïve and negative selection pools that overlap with the positiveselection pool are of very short length. However, this type of potentialbias can be identified by comparing the abundance of given sequences inthe positive pool to the negative pool. It is possible that shortersequences were preferentially amplified during the PCR reaction used torecover the DNA that was subsequently submitted for Illumina sequencing.In addition, a previous analysis of the naïve Fab library diddemonstrate that there was a bias towards shorter CDR H3 loop lengths,which was likely attributable to differences in the efficiency of thelibrary mutagenesis reaction with oligos of different lengths. Thisissue may be addressed by comparison of the length distribution of thehypervariable regions sequenced by traditional Sanger methods to thosesequenced in the deep sequencing analysis.

A limiting step to molecular display technologies is the need forcorrectly folded, purified antigen. For example, multi-domain membranerepresent more than 70% of current drug targets due to their role in theprogression and tumorigenesis of numerous cancers [1], yet theproperties of these proteins makes their production and purificationextremely difficult. The instability of membrane proteins also makesthem challenging targets to work with during in vitro libraryselections, as many of these proteins depend on the membrane environmentfor their correct structure and function. The methodology reported herebypasses the need for purified antigen and allows library selectiondirectly to cell-surface targets. Consequently, the CellectSeqmethodology increases the likelihood that the selected antibodies willrecognize epitopes on the native, functionally relevant structure of thetarget antigen. The ability to select for specifically binding phageclones without the need for purified antigen will significantly expandthe range of antigens that can be targeted using phage displaytechnology.

The described methods could also be tailored to the specific needs ofthe antigen of interest. For instance, the CellectSeq approach can becombined with protocols that involve screening libraries against cellsin the presence of ligands, with the goal of targeting active forms ofreceptors [6]. In cases in which the target of interest may be a memberof an oligomeric complex, the selection can be performed using cellsco-transfected to express all of the relevant complex members, with theintention of isolating antibodies specific to the multimerized protein.One example of relevance here is Her2, as it known to homo- andhetero-dimerize with the other members of the EGFR family [27].

Example 2. PCR-Based Recovery of Antibody Fragments to AdditionalCell-Surface Targets

The rescue strategies described herein make use of both the unique H3and L3 CDR sequences.

As an alternative to identifying positive Fabs by clonal cell ELISAs,two different PCR based recovery methods are used (FIG. 6A, 6B). Asdepicted in FIG. 6A, two primer sets specific for both CDR H3 and L3 areused to make the recovery more specific. Primers are designed to annealto the L3 and H3, and amplify two fragments, in both directions. Thisresults in two fragments that both contain the L3 and H3 regions. Thetwo fragments can be annealed, and then a single round of DNA extensionis done. The resulting product can then be ligated and transformed intoE. coli to recover the desired phage clone in the original librarydisplay vector. As depicted in FIG. 6B, three primer sets are used toamplify three fragments, in a strategy that makes use of both the H3 andL3 unique sequences and unique Nsi1 and Nhe1 sites in the library phagevector. The three fragments are annealed, extended by PCR, and subclonedinto an IPTG inducible protein expression vector with compatible Nsi1and Nhe1 restriction enzyme sites. The rescued Fab can be expresseddirectly from the resulting vector.

The phage-Fab clones that were rescued from the positive selection poolare shown in FIG. 9 . Listed are the phage-Fab clones targeting variouscell surface receptors that were successfully rescued from the positiveselection pool. The following is displayed: the cell line used toexpress the antigen of interest; the phage-display library used forselection (F: Fab, G:scFv); the rescue strategy used to recover theclones (1.Clonal ELISA, 2. cdrH3 PCR, 3.cdrL3:cdrH3 PCR); the rank ofthe sequence in the round four positive selection pool based on rawcounts which reflects the number of times the sequence was observed inthe pool; the CDR L3 and H3 sequences obtained from the round fourpositive selection output; the raw counts and percentage those countsrepresent of the entire output pool for the round four and round threepositive and negative pools; whether the rescued clones have beenvalidated for cell binding. The conditions used for positive andnegative selections are also annotated. Note that GUP is a cocktail ofGlucosamine, Uridine and PugNAc.

Example 3. Rapid Isolation of Antibody Fragments Specific to Cell GrowthConditions

FIG. 7 depicts a flow chart of the selection strategy used to isolateFab clones specific for cell surface O-GlcNAc-dependent epitopes,demonstrating that the CellectSeq method can be used to isolate Fabsspecific to cell growth conditions. The positive selection begins apre-absorption step in which the library phage are incubated with MCF7breast cancer cells grown in DMEM (Dulbecco's Modified Eagle Medium)(high glucose version) supplemented with 10% FBS. After incubation, themixture is pelleted to remove the library clones bound to the cells.These clones are likely specific for cell-surface epitopes that are notof interest, or are non-specific binding clones. The library phageremaining in the supernatant are incubated with the MCF7 cells grown inDMEM (high glucose version) supplemented with 10% FBS plus 30 mMGlucosamine (G), 5 mM Uridine (U) and 50 μM PugNAc (P) (collectivelyreferred to as GUP), non-binding phage are then washed away, and thephage bound to the GUP treated MCF7 cells are amplified in an E. colihost. The amplified phage are then purified and used in the next roundof selection. In parallel, the negative selection is carried out byincubating library phage with MCF7 cells that have been grown in theabsence of GUP treatment. Phage clones that do not bind to the cells arewashed away, and the remaining bound phage are amplified in an E. colihost for the next round of selection. O-GlcNAc enrichment is achieved byadding GUP (a cocktail of Glucosamine, Uridine and PugNAc).

FIG. 8 depicts an ELISA graph of binders chosen from R40 from theselection strategy used to isolated Fabs specific for surfaceO-GlcNAc-dependent epitopes. B4 binder ranked #1 with ratio of 1.8, A4binder ranked #2 with ratio of 1.6, C4 binder ranked #4 with ratio of1.5 and C3 binder ranked #7 with ratio 1.5.

Example 4. Deep Sequencing to Decode Variable Regions of AffinityReagents

FIG. 10 provides diagrams for some deep/high-throughput sequencingstrategies used to decode variable regions of affinity reagents in apositive or negative selection pool. In the examples shown here, one ormore complementarity determining regions (CDRs) of synthetic antibodiesare decoded by deep sequencing.

Materials and Methods

Positive and negative selection pool phages from rounds three and fourwere infected into XL1Blue cells and grown overnight in 2YT supplementedwith 100 ug/ml carbenicillin. Cultures were miniprepped to obtainphagemid DNA and normalized to 25 ng/ul to use as templates for PCR. PCRprimers added barcodes and platform-specific adapters, while amplifyingone or more variable regions of the affinity reagent by annealing toadjacent regions of the affinity reagent framework.

Strategy 1: IIlumina Sequencing of CDRs L3 and H3

The forward PCR primer was composed of a paired-end compatible Illuminaadaptor sequence (5′AATGATACGGCGACCACCGAGATCT-3′) (SEQ ID NO: 223) andan annealing site upstream of CDR-L3 (5′GCAGCCGGAAGACTTCGCAACTTATTACTGTCAGC-3′) (SEQ ID NO: 224). The reversePCR primer was composed of a paired-end compatible Illumina adaptorsequence (5′ CAAGCAGAAGACGGCATACGAGAT-3′) (SEQ ID NO: 225), a five basebarcode (5′NNNNN-3) (SEQ ID NO: 226), and an annealing site downstreamof CDR-H3 (5′GGTGACCAGGGTTCCTTGACCCCAGTAGTC-3′) (SEQ ID NO: 227).

PCR reactions were performed with the high fidelity polymerase ExTaq(TaKaRa) and 400 ng of template phagemid DNA. Reactions were subjectedto one denaturation step for 30 sec at 95° C., followed by 14 cycles of30 sec at 94° C. and 60 sec at 72° C., with a final extension for 5 minat 72° C. PCR products were cleaned enzymatically with ExoI to removeresidual primers, SAP to dephosphorylate dNTPs and Dpn1 to digestmethylated phagemid template DNA. PCR products were quantitated usingdsDNA-specific fluorescent dye (PicoGreen), normalized, pooled andpurified by gel extraction of the correct fragment size (1007 bp).

The purified DNA fragments were subjected to Illumina DNA sequencing onGAIIx or HiSeq platforms, using custom read primers and read lengths:Read 1 forward (L3) primer (5′ CAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAA-3′)(SEQ ID NO: 228) for a minimum of 30 bases; Read 2 forward (barcode)primer (5′ GACTACTGGGGTCAAGGAACCCTGGTCACC-3′) (SEQ ID NO: 229) for aminimum of 5 bases; Read 3 reverse (H3) (5′GGTGACCAGGGTTCCTTGACCCCAGTAGTC-3′) (SEQ ID NO: 230) for a minimum of 65bases. Each sequencing read was assigned to its correct pool of thebasis of its unique barcode sequence. The reads were filtered accordingto their Phred score [31]. Briefly, all sequences with phred scores of20 or higher for every base were kept. DNA sequences were translated todecode the sequences of CDRs L3 and H3.

Strategy 2: Illumina Sequencing of CDRs L3 and H3, with OptionalSequencing of H2 and H1

The forward PCR primer was composed of a paired end Read 1 Illuminaadaptor sequence(5′AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3) (SEQ IDNO: 231), barcode (5′NNNNNNNN-3′) (SEQ ID NO: 232) and an annealing sitedownstream of CDR-H3 (5′GGTGACCAGGGTTCCTTGACCCCAGTAGTC-3′) (SEQ ID NO:233). The reverse PCR primer was composed of a paired end Read 2Illumina adaptor sequence (5′ CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3′)(SEQ ID NO: 234), optional barcode (5′NNNNNN-3′), and an annealing siteupstream of CDR-L3 (5′ CAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAA-3′) (SEQ IDNO: 235). PCR reactions were carried out using ExTaq, as described forStrategy 1.

The purified DNA fragments were subjected to Illumina DNA sequencing onGAIIx, HiSeq or Miseq platforms, using standard paired end read primersand 2×150 bp read lengths or longer, to span CDR-H2 and CDR-H1 inaddition to barcode and CDR-H3 (read 1) or CDR-L3 (read 2).

Strategy 3: IonTorrent Sequencing of CDR H3

The forward PCR primer was composed of an IonTorrent Adapter A sequence(5′ CCATCTCATCCCTGCGTGTCTCCGACTCAG-3′) (SEQ ID NO: 236), barcode (5′NNNNNNNNNC-3′) (SEQ ID NO: 236) and an annealing site upstream of CDR-H3(5′AGGACACTGCCGTCTATTAT-3′) (SEQ ID NO: 237). The reverse PCR primer wascomposed of IonTorrent adapter P1 sequence (5′CCTCTCTATGGGCAGTCGGTGAT-3′) (SEQ ID NO: 238) and an annealing sitedownstream of CDR-H3 (5′AGGACACTGCCGTCTATTAT-3′) (SEQ ID NO: 239). PCRreactions were carried out using Phusion with one denaturation step at98 C for 5 min, followed by 14 cycles of 5 sec at 98° C., 10 sec at 54°C., 15 sec at 72° C., with a final extension for 10 min at 72° C.Residual primers and dNTPs were removed using column (Qiagen), and PCRproducts were quantitated, normalized and pooled, for single endsequencing on an IonTorrent platform.

Although preferred embodiments of the invention have been describedherein, it will be understood by those skilled in the art thatvariations may be made thereto without departing from the spirit of theinvention or the scope of the appended claims. All references citedherein, including those in the attached reference list, are incorporatedby reference.

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What is claimed is:
 1. A method of identifying a potential binder to acell surface antigen, the method comprising: (a) providing a displaylibrary comprising clones, each of which displaying an antibody orantibody fragment, wherein the antibody or antibody fragment has anantibody variable region comprising: CDR L1 comprising an amino acidsequence identified as SEQ ID NO: 256; CDR L2 comprising an amino acidsequence identified as SEQ ID NO: 257; CDR L3 comprising an amino acidsequence identified as SEQ ID NO: 258; CDR H1 comprising an amino acidsequence identified as SEQ ID NO: 259; CDR H2 comprising an amino acidsequence identified as SEQ ID NO: 260; and CDR H3 comprising an aminoacid sequence identified as SEQ ID NO: 261; wherein each of the clonescomprises DNA encoding the antibody variable region displayed thereby;(b) screening the library against a population of cells that express thecell surface antigen to produce a positive selection pool of libraryclones and against a population of cells for producing a negativeselection pool of library clones; (c) sequencing DNAs encoding antibodyvariable regions of the positive selection pool and of the negativeselection pool; and (d) identifying, among the sequenced DNAs, DNAencoding an antibody variable region that is more abundant in thepositive selection pool than in the negative selection pool, therebyidentifying an antibody variable region that is more abundant in thepositive selection pool than in the negative selection pool, therebyidentifying a potential binder to the cell surface antigen.
 2. Themethod of claim 1, further comprising a step of producing a purifiedantibody or antibody fragment, wherein the purified antibody or antibodyfragment comprises the antibody variable region encoded by the DNAidentified to be more abundant in the positive selection pool than inthe negative selection pool.
 3. The method of claim 1, wherein thedisplay library is a phage-display library and the clones are phageclones.
 4. The method of claim 1, wherein the antibody or antibodyfragment is a Fab or a scFv.
 5. The method of claim 1, wherein the cellsurface antigen is a protein, wherein the cells that express the cellsurface antigen are cells of a cell line which are transfected toexpress the cell surface antigen, and wherein the cells for producingthe negative selection pool are cells of the cell line which are nottransfected to express the cell surface antigen.
 6. The method of claim1, wherein the sequencing is done by deep sequencing.
 7. The method ofclaim 1, wherein step (b) comprises multiple rounds of screening thelibrary against the population of cells that express the cell surfaceantigen and against the population of cells for producing a negativeselection pool of library clones.
 8. The method of claim 1, wherein step(d) comprises identifying DNA encoding an antibody variable region thatis more abundant in the positive selection pool than in the negativeselection pool by a factor selected from the group consisting of afactor of at least 2, a factor of at least 3, a factor of at least 4 anda factor of at least
 5. 9. The method of claim 1, wherein the cellsurface antigen is CD133 or ErbB3.
 10. A method of identifying a binderto a cell surface antigen, the method comprising: (a) providing adisplay library comprising clones, each of which displaying an antibodyor antibody fragment, wherein the antibody or antibody fragment has anantibody variable region comprising: CDR L1 comprising an amino acidsequence identified as SEQ ID NO: 256; CDR L2 comprising an amino acidsequence identified as SEQ ID NO: 257; CDR L3 comprising an amino acidsequence identified as SEQ ID NO: 258; CDR H1 comprising an amino acidsequence identified as SEQ ID NO: 259; CDR H2 comprising an amino acidsequence identified as SEQ ID NO: 260; and CDR H3 comprising an aminoacid sequence identified as SEQ ID NO: 261; wherein each of the clonescomprises DNA encoding the antibody variable region displayed thereby;(b) screening the library against a population of cells that express thecell surface antigen to produce a positive selection pool of libraryclones and against a population of cells for producing a negativeselection pool of library clones; (c) sequencing DNAs encoding antibodyvariable regions of the positive selection pool and of the negativeselection pool; (d) identifying, among the sequenced DNAs, DNA encodingan antibody variable region that is more abundant in the positiveselection pool than in the negative selection pool; and (e) producing apurified antibody or antibody fragment, wherein the purified antibody orantibody fragment comprises the antibody variable region encoded by theDNA identified to be more abundant in the positive selection pool thanin the negative selection pool, and confirming that the purifiedantibody or antibody fragment binds to the cell surface antigen inpurified form and/or binds to cells expressing the cell surface antigen,thereby identifying a binder to the cell surface antigen.
 11. The methodof claim 10, wherein the display library is a phage-display library andthe clones are phage clones.
 12. The method of claim 10, wherein theantibody or antibody fragment is a Fab or a scFv.
 13. The method ofclaim 10, wherein the cell surface antigen is a protein, wherein thecells that express the cell surface antigen are cells of a cell linewhich are transfected to express the cell surface antigen, and whereinthe cells for producing the negative selection pool are cells of thecell line which are not transfected to express the cell surface antigen.14. The method of claim 10, wherein the sequencing is done by deepsequencing.
 15. The method of claim 10, wherein step (b) comprisesmultiple rounds of screening the library against the population of cellsthat express the cell surface antigen and against the population ofcells for producing a negative selection pool of library clones.
 16. Themethod of claim 10, wherein step (d) comprises identifying DNA encodingan antibody variable region that is more abundant in the positiveselection pool than in the negative selection pool by a factor selectedfrom the group consisting of a factor of at least 2, a factor of atleast 3, a factor of at least 4 and a factor of at least
 5. 17. Themethod of claim 10, wherein the cell surface antigen is CD133 or ErbB3.18. The method of claim 1, wherein the CDR-L3 comprises an isoleucineresidue flanking the C-terminal amino acid residue thereof.
 19. Themethod of claim 10, wherein the CDR-L3 comprises an isoleucine residueflanking the C-terminal amino acid residue thereof.